Method of obtaining intrinsic factor preparations of enhanced potency



United States Patent 0 cc METHOD OF QETAINING INTRINSIC FACTOR PREPARATIONS 0F ENHANCED POTENCY Kenneth C. Robbins, Chicago, 11]., assignor to Armour and Company, Chicago, Ill.,.a corporation of Illinois No Drawing. Application September 18, 1952, Serial No. 310,372

3 Claims. (Cl. 167,74)

This invention relates to a method of obtaining intrinsic factor preparations of enhanced potency and preparations obtained thereby. The method of this invention has particular utility in producing intrinsic factor concentrates from hog duodenum, but it can also be advantageously applied to hogstomach, and other intrinsic factor-bearing mammalian tissues.

It is known that at least duodenum and stomach tissues of hogs contain a substance which is of value in the treatment of anemia in humans, and particularly in the treatment of pernicious anemia. The value of this substance can be most dramatically demonstrated for pernicious anemia patients in relapse, and the efficacy of a particular vhog duodenum or stomach preparation in treating such .patients has been accepted as the most reliable method for assaying the potency of the preparation.

The substance referred to above is generally designated as the intrinsic factor or as Oastles intrinsic factor. However, the intrinsic factor has not been isolated, and therefore its exact nature is still unknown. For the purpose of this application the term intrinsic factor will be used to designate the component (or components) in-hog duodenal or stomach tissue which is of value in the treatment of anemic conditions, particularly of the macrocytic type.

Heretofore various intrinsic factor preparations have been utilized in the treatment of pernicious anemia, but all. of them have suffered from the defects of requiring the patient to swallow objectionably large quantities of unpleasant material. For example, the average daily re quirement of whole hog duodenum by a pernicious anemia patient is about one-fourth to one-half pound, and even when desiccated at least 20 to 40 grams are required. Efforts have been made to improve this situation by attempting to prepare more concentrated intrinsic factor preparations, and this has resulted in preparations which are adequate in daily quantities of as low as 2 grams. However, when the previously known concentrates are employed in less than 1 gram per day doses on pernicious anemia patients in relapse, a full response is not obtained. Daily doses of the order of 1 to 2 grams are objectionable from the standpoint of the patient. For example, to administer 1 gram of material, about 5 tablets or capsules are required. The recognized objective has been to prepare arconcentrate which would give a full response by the intake of one small tablet per day, that is, of the order of 200 milligrams or less, but until now this has not been achieved in spite of considerable efforts towards solving the problem.

It is therefore a general object of this invention to prepare intrinsic factor preparations of enhanced potency. Amore specific object is to prepare intrinsic factor preparations which are sufliciently concentrated that a perni- 2,770,570 Patented Nov. 13, 1956 cious anemia patient in relapse need only take one small tablet per day to obtain a full response. Further objects and advantages will appear as the specification proceeds.

The preferred starting material for use in the process of this invention is whole hog duodenum, but other intrinsic factor-bearing tissue, such as hog stomach tissue, etc., can also be employed. The following discussion will be concerned primarily with the processing of hog duodenurn tissue, but it will be understood to apply to other intrinsic factor-bearing tissue.

Preparatory to extracting the intrinsic factor from the duodenal tissue, it is preferred to comminute the tissue by hashing, grinding, etc. if desired, the fresh hog duodenum can be frozen and then ground in the presence of Dry Ice to prevent any possibility of loss of activity. However, this technique is not essential.

The comminuted duodenal tissue is then extracted with water to obtain .an aqueous extract containing the intrinsic factor. The volume of water employed is not particularly critical, but a convenient range is from 2 to 4vol umes of water to each volume of duodenal tissue. It is preferred to carry out the aqueous extraction at an acidic pH not substantially below pH- 2. Optimum results seem to be obtained at a pH from about 3 to 4. The extraction can be carried out by any of the usual techniques such as agitating the suspension of tissue .in the water by a motor-driven stirrer, etc. In order to prevent loss of activity it is believed desirable to keep the temperature below 15 C., although somewhat higher temperatures canprobably be employed. Excellent results are obtained at temperatures below 5 C.

The aqueous extract of the intrinsic factor (or supernatant) is separated from the tissue residue by any suitable method such as filtration, centrifugation, etc. The aqueous extract is next fractionated with ethanol to obtain as a precipitate an intrinsic factor concentrate. This step is believed to be largely responsible for the greatly improved results of the process of this .invention. Instead of ethanol other liquid organic precipitating agents can be employed such as the aliphatic monohydric alcohols having less than five carbon atoms and acetone. Other lower aliphatic ketones, such as methyl ethyl ketone, can be substituted for acetone. Methanol is an especially desirable substitute for ethanol.

The ethanol or other similar organic precipitating agent is added to the aqueous extract until the ethanol concentration of the extract is between 20 to 60% by volume.

Preferably, the fractionation should be carried out at an ethanol concentration in the extract of between 30 to 50% by volume, and optimum results appear to be obtained at concentrations of between 35 to 45%.. The pH for the fractionation is somewhat critical, and is related to the preferred pH for the extraction. As indicated above, the extraction is preferably carried out at a pH not over 4 (that is, from pH 2 to pH 4) and this is related to the preferred pH for the fractionation, which is preferably above 4, and specifically from pH 4 to 6. Optimum results are achievedfor the fractionation at pHs from 4.5 to 5.0. However, fair results can be obtained outside of this range. Because of the presence of the relatively high concentrations of organic solvent in the extract during the fractionation step, it is preferred to keep the temperature of the extract below 0 C., and preferably below -3 C. Excellent results are obtained at temperatures of around 5 C. with alcohol concentrations up to 40% by volume, although at higher alcohol concentrations it may be desirable to use a somewhat lower temperature in order to prevent denaturation of the intrinsic factor.

The result of the above fractionation step is that a precipitate forms which consists of an intrinsic factor preparation of enhanced potency, and in fact, of higher potency than any preparation which has heretofore been known. The intrinsic factor containing precipitate is separated from the supernatant (filtration, centrifugation, etc.) and is prepared for medicinal use. For example, the separated precipitate can be removed by centrifugation, and is dissolved in 3 volumes of ice cold water. The solution is clarified and lyophilized.

The pH adjustment in the extraction step can be made with hydrochloric acid or other suitable acids, such as sulphuric, phosphoric, lactic, citric, etc. The pH adjustment in the fractionation step can be made with sodium hydroxide or other alkali metal hydroxides or an alkaline earth metal hydroxide or carbonate such as potassium hydroxide, calcium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, etc.

In regard to the fractionation step, one preferred procedure is to first adjust the pH of the aqueous extract following its separation from the residue to a pH between 4 and 6 prior to the addition of the ethanol or other organic precipitating agent. The pH adjustment will usually cause a small precipitate to form, which is very low in intrinsic factor content. Therefore, it is preferred to remove this precipitate prior to the addition of the organic precipitating agent. Following the removal of the low potency precipitate, the ethanol is added to the solution to a concentration of between 35 to 45% by volume, thereby obtaining as a precipitate the desired intrinsic factor concentrate.

To more fully .illustrate this invention, it is desired to set forth the following detailed examples.

Example I The following steps are recommended for isolating the intrinsic factor of Castle from hog duodenum:

(1) Suspend 1 volume of fresh frozen comminuted hog duodenum in 3 volumes of ice cold distilled water.

(2) Adjust the pH of the suspension to 3.5 :1 with 1N HCl.

(3) Agitate the suspension for 2 hours at about 1 C.

(4) Remove the residue from the extract by centrifugation at about 1 C. and discard it.

(5) Adjust the pH of the supernatant to 4.7 id with 1N NaOH, and separate the resulting low potency precipitate by filtration.

(6) Bring the solution to 40% ethanol concentration at -5 C. with 95% ethanol and hold overnight at -5 C.

(7) Remove the precipitate by centrifugation.

(8) Dissolve the precipitate in 3 volumes of ice cold water, clarify, and lyophilize.

The final product obtained by the above procedure will be an intrinsic factor preparation of enhanced potency.

Example II The following constitutes an actual experimental example. 77.25 kg. of hog duodenal tissue were suspended in 230 liters of distilled water at a pH of 3.5 (H01) and an extraction was carried out at 1 C. for two hours. The extract was separated from the residue by centrifugation at 1 C. The extract having a volume of 200 liters was then adjusted to pH 4.7 (NaOH) and held overnight at 1 C. The precipitate thus formed was separated from the supernatant by filtration at 1 C. and discarded. The supernatant (140 liters) was brought to 40% ethanol by adding 95% ethanol thereto and held overnight at 5 C. The precipitate was separated by centrifugation at -5 C., and the supernatant was discarded. The precipitate thus obtained weighed 920 grams. This precipitate was further processed by dissolving it in 4 liters of ice cold water. The solution was clarified and lyophilized. The lyophilized preparation having a weight of 142 grams was found to be a high potency concentrate of the intrinsic factor of Castle by administering portions thereof to pernicious anemia patients in relapse.

In one series of tests with the product obtained above, the intrinsic factor concentrate was formulated into tablets and capsules in admixture with vitamin B12. One pernicious anemia patient in relapse received 600 milligrams of the intrinsic factor concentrate prepared above plus 8 micrograms of B12 per day, and evidenced a full U. S. P. response. Another patient showed a full response on a daily intake of 300 milligrams of the intrinsic factor concentrate together with 8 micrograms of B12. Still another patient showed a full response on a daily intake of milligrams of the intrinsic factor concentrate plus 3.0 micrograms of B12.

In other words, the above tests indicate that a full U. S. P. response could be expected from pernicious anemia patients in relapse by giving them one small tablet or capsule per day containing as little as 120 milligrams of the intrinsic factor concentrate prepared by the procedure set out in this example, together With30 micrograms of vitamin B12.

While in the foregoing specification this invention has been set out in considerable detail for purpose of illustration, it will be apparent to those skilled in the art that many of the details set forth can be varied widely without departing from the spirit of the inveniton.

I claim:

1. In a process for preparing an intrinsic factor preparation of enhanced potency, the sequence of steps comprising extracting intrinsic factor-bearing tissue selected from the group consisting of hog stomach tissue and hog duodenum tissue with water at an acidic pH between 2.0 and 4.0 to obtain an intrinsic factor extract, adjusting the pH of said extract to a pH from 4.5 to 5.0 to form a precipitate of relatively low intrinsic factor potency, separating the precipitate from the supernatant, and fractionating said supernatant to obtain a precipitate of enhanced intrinsic factor potency, said fractionating step being carried out by adding an alcohol to said supernatant until the alcohol concentration thereof is from 30 to 50% by volume while maintaining the pH of said extract within the range from pH 4.0 to 6.0, said alcohol being selected from the group consisting of methanol and ethanol.

2. In a process for preparing an intrinsic factor preparation of enhanced potency, the sequence of steps comprising extracting intrinsic factor-bearing tissue selected from the group consisting of hog stomach tissue and hog duodenum tissue with water at an acidic pH between 3.0 and 4.0 to obtain an intrinsic factor extract, adjusting the pH of said extract to a pH from 4.5 to 5 .0 to form a precipitate of relatively low intrinsic factor potency, separating the precipitate thus formed from the supernatant, and then fractionating said supernatant to obtain a precipitate of enhanced intrinsic factor'potency, said fractionating step being carried out by adding ethanol to said supernatant until the ethanol concentration thereof is from 35 to 45% by volume While maintaining the pH of said supernatant within the range from 4.5 to 5.0.

3. In a process for preparing an intrinsic factor preparation of enhanced potency, the sequence of steps comprising extracting intrinsic factor-bearing tissue selected from the group consisting of hog stomach tissue and hog duodenum tissue with water at an acidic pH between 2.0 and 4.0 to obtain an intrinsic factor extract, raising the pH of said extract to a pH above 4.0 and below 6.0 to form a precipitate of relatively low intrinsic factor potency, separating the precipitate thus formed from the supernatant, then fractionating said supernatant to obtain a precipitate of enhanced intrinsic factor potency, said fractionating step being carried out by adding an alcohol to said extract until the alcohol concentration thereof is from 30 to 50% by volume While maintaining the pH of said extract Within the range from 4.0 to 6.0, said alcohol being selected from the group consisting of methanol and ethanol.

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Glass: Blood, v01. 8, No. 18, October 1953, pages 867 and 868.

Prusoif: Am. Chem. Soc., abst. of papers, 8th meeting, 5 1950, p. 2721.

Ternberg et al.: J. A. C. 8., vol. 71, November 1949, page 3858.

1,813,788 Walden July 7, 1931 1,829,270 Fogelson Oct. 27, 1931 10 2,477,541 Ivy et a1. July 26, 194-9 

1. IN A PROCESS FOR PREPARING AN INTRINSIC FACTOR PREPARATION OF ENHANCED POTENCY, THE SEQUENCE OF STEPS COMPRISING EXTRACTING INTRINSIC FACTOR-BEARING TISSUE SELECTED FROM THE GROUP CONSISTING OF HOG STOMACH TISSUE AND HOG DUODENUM TISSUE WITH WATER AT AN ACID PH BETWEEN 2.0 AND 4.0 TO OBTAIN AN INTRINSIC FACTOR EXTRACT, ADJUSTING THE PH OF SAID EXTRACT TO A PH FROM 4.5 TO 5.0 TO FORM A PRECIPITATE OF RELATIVELY LOW INTRINSIC FACTOR POTENCY, SEPERATING THE PRECIPITATE FROM THE SUPERNATANT, AND FRACTIONATING SAID SUPERNATANT TO OBTAIN A PRECIPITATE OF ENHANCED INTRINSIC FACTOR POTENCY, SAID FRACTIONATING STEP BEING CARRIED OUT BY ADDING AN ALCOHOL TO SAID SUPERNATANT UNTIL THE ALCOHOL CONCENTRATION THEREOF IS FROM 30 TO 50% BY VOLUME WHILE MAINTAINING THE PH OF SAID EXTRACT WITHIN THE RANGE PH 4.0 TO 6.0, SAID ALCOHOL BEING SELECTED FROM THE GROUP CONSISTING OF METHANOL AND ETHANOL. 